14-3-3 proteins interact with specific MEK kinases.
MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.
Fanger
G R
GR
Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Widmann
C
C
Porter
A C
AC
Sather
S
S
Johnson
G L
GL
Vaillancourt
R R
RR
D048948:14-3-3 Proteins; D000818:Animals; D019556:COS Cells; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases